Double-strand breaks (DSBs) in DNA occur as a result of environmental challenges, such as exposure to ionizing radiation (IR) or during normal cell metabolism, such as DNA replication. In heterochromatin, DSBs are a major threat to genome stability, since the abundance of repetitive sequences maximizes the potential for aberrant recombination and genome instability during repair. However, the regulation of repair processes operating in this large chromatin domain is mostly unknown. The Drosophila model system is ideal for studying heterochromatin DSB response. It features genetic tractability comparable to yeast, complex heterochromatin similar to mammals, and is advantageous for cytological approaches because all pericentromeric regions of different chromosomes are concentrated in one distinct nuclear domain. Our previous studies with this model system revealed that heterochromatin responds dynamically to DSBs: the entire domain expands and the damaged sites move to outside the domain to complete homologous recombination (HR) repair. Similar responses in mammalian cells suggest that this pathway is highly conserved. While early HR processing of DSBs occurs within the heterochromatin domain, later HR steps are postponed until relocalization is complete. Loss of heterochromatin components results in defective relocalization of repair centers, aberrant recombination and chromosome rearrangements. These results reveal the importance of heterochromatin proteins in coordinating the spatial and temporal dynamics of HR repair in heterochromatin and in protecting repeated DNA sequences from genome instability. To significantly advance our understanding of this important and novel mechanism, we will combine multi-disciplinary approaches to identify pro-/anti-recombinases and nuclear architecture components required for successful HR repair of heterochromatic DSBs. These studies will uncover the mechanisms that normal cells use to protect repeats from environmental mutagens. In addition, this research will contribute to our understanding of the mechanisms that generate chromosome rearrangements when mutations or environmental challenges inactivate the safeguarding mechanisms. This knowledge is expected to contribute to the future development of tools for prevention, diagnosis, and treatment of human diseases associated with repeated DNA instability, such as cancer and birth defects.